Conclusions These findings lend support to the novel concept that factors in addition to absolute weight burden, such as qualitative features of adipose tissue, might be important determinants of cardiovascular disease. Therapeutic modulation of the adipose phenotype might represent a target for treatment in obesity.

There is growing recognition that many overweight individuals maintain a relatively favorable cardiometabolic profile even in extreme obesity. Determinants of a metabolically healthier obese phenotype are poorly understood and likely multifactorial, relating to differences in body fat distribution, physical activity, and adipose metabolism (1). In addition, recent data suggest that inflammation of adipose tissue orchestrated by monocyte/macrophage infiltration and overproduction of proatherogenic cytokines might mediate metabolic and vascular disease in human obesity (2,3). Chronic activation of the immune system has been strongly implicated in the pathogenesis of obesity-associated disorders, including type 2 diabetes mellitus, cancer, and cardiovascular disease, and is a growing target of interest for therapeutic intervention (4).

We and others have previously shown that pro-inflammatory changes in fat are linked to metabolic stress and endothelial dysfunction (2,5,6). The goals of the present study were to determine whether qualitative differences exist between the adipose tissue of obese versus normal weight individuals and to investigate whether overweight patients with reduced adipose inflammation are polarized toward a lean phenotype.

Methods

Study subjects

We enrolled overweight adult men and women (≥18 years of age) with a body mass index (BMI) of ≥25 kg/m2 receiving care at the Boston Medical Center. We also recruited lean adults (BMI <25 kg/m2) through advertisements to the general public. Subjects with unstable medical conditions or pregnancy were excluded. The study was approved by Boston Medical Center Institutional Review Board, and all subjects gave written informed consent. Blood pressure, heart rate, height, weight, BMI, and waist circumference (WC) were recorded for each subject, and all biochemical analyses were quantified from fasting blood samples.

Subcutaneous adipose tissue collection

From each subject, we collected abdominal subcutaneous adipose tissue via percutaneous needle biopsy technique or directly harvested during gastric bypass surgery, as previously described (2,7). All subjects were in a fasting state for ≥12 h before biopsy. Each subject provided a single biopsy specimen from the subcutaneous region for analysis.

Vascular studies

For each subject, brachial artery ultrasound studies were performed in a fasting state. Brachial vasomotor responses were examined with a noninvasive, standardized method of ultrasound with a Toshiba Powervision 6000 system (Toshiba, Tokyo, Japan), as previously described (2,11). Flow-mediated dilation (FMD) after a 5-min cuff occlusion in an upper arm position and nitroglycerin-mediated dilation of the brachial artery served as measures of endothelium-dependent and -independent dilation, respectively. Sublingual nitroglycerin (0.4 mg) was omitted if contraindicated or the subject declined.

Statistical analysis

Analyses were completed with SAS for Windows (version 9.1, SAS Institute, Inc., Cary, North Carolina). Data are presented as mean ± SD, median with interquartile range, or proportions (%). Categorical group differences were examined with the chi-square test or Fisher exact test as appropriate. Kolgomorov-Smirnov tests, histograms, and normal probability plots were used to determine whether continuous variables were normally distributed or skewed. Natural log transformation was applied only to continuous variables not meeting normality, which specifically were glucose, homeostasis model assessment (HOMA), insulin, triglycerides, as well as adipose expression of interleukin (IL)-8, catalase, peroxiredoxin-1, guanylate cyclase I alpha/beta, IL-6, and IL-1beta. The latter 4 genes did not reach normality despite transformation and were analyzed by nonparametric methods. Group differences for continuous variables were examined with analysis of variance with Tukey post-hoc analysis, and the Kruskal-Wallis test was used for skewed variables. Univariate associations between vascular parameters or HOMA and clinical data were examined in obese subjects with Pearson's correlation. Alternatively, Spearman's rank correlation was used for skewed data. Multiple linear regression was used to determine whether CLS status was independently associated with FMD. Univariate clinical correlates of FMD with significance level of p < 0.1 were included in the model. For all analyses, p value <0.05 was considered statistically significant.

Results

Clinical and histological data

A total of 109 obese subjects (mean age 42 ± 11 years, 86% women) and 17 lean individuals (mean age 33 ± 12 years, 77% women) completed this study. The clinical characteristics of all participants are displayed in Table 1. The majority of obese individuals (72%) demonstrated evidence of adipose inflammation characterized by tissue presence of macrophage crown-like structures in subcutaneous fat (CLS+, n = 78) as shown in Figure 1, which were absent in 28% of overweight subjects (CLS−, n = 31). In contrast, all lean subjects were noninflamed (CLS−). As expected, the lean group had significantly lower BMI, WC, plasma insulin, HOMA, LDL-C, glycosylated hemoglobin A1C (HbA1c), triglycerides, glucose, hs-CRP, hypertension, diabetes prevalence, medication use, and higher HDL-C levels compared with the CLS+ obese group (p < 0.05). However, despite the same degree of adiposity, sex distribution, and age range as the CLS+ group, insulin levels and HOMA were significantly lower in CLS− obese, exhibiting values that were intermediate to the lean and inflamed overweight groups (p < 0.05).

Discussion

In the present study, we demonstrate that obese individuals display more pro-atherogenic vascular, metabolic, and adipose tissue profiles as compared with lean subjects. The key finding was that, for the same degree of severe obesity, individuals with reduced adipose inflammation exhibited an “intermediate” clinical phenotype with arterial function similar to normal weight subjects. We observed parallel trends in adipocytokine expression that mirrored systemic profiles, suggesting a biological connection. In this regard, the findings prompt speculation that aspects of cardiovascular disease mechanisms might have origins within the adipose microenvironment.

Animal models consistently show that excess adiposity induces a chronic state of immune system activation, characterized by adipose tissue influx of macrophages, neutrophils, and T lymphocytes that stimulate adipocytokine production implicated in the temporal development of insulin resistance (13). We now recognize that the consequence of adipose inflammation likely extends beyond metabolic disturbance to cause vascular injury and atherosclerosis. For example, adipose tissue inflammation induces endothelial activation and alters blood flow in the microcirculation of obese mice (14). Inflamed perivascular fat preferentially localizes to atherosclerotic regions, and adipocytokines impair vasomotor function (15). These findings show that functional properties of blood vessels are adversely modulated by the state of the adipose microenvironment. Thus, it is plausible to speculate that disease at the adipose level might extend and reflect in the systemic vasculature, because this concept is supported by our finding that obese patients with reduced adipose inflammation displayed favorable arterial responses as in lean subjects. This might hold clinical significance, because prospective studies consistently show that endothelial dysfunction predicts cardiovascular events (16).

The degree of adipose immune activation in humans is more variable than in genetically modified experimental animals, and this heterogeneity in tissue phenotype provides a window of opportunity to investigate how adipose changes relate to clinical disease. In this regard, we demonstrated that a specific pattern of macrophage build-up in fat is associated with metabolic and endothelial dysfunction (2,7). The key issue of what sets off the inflammatory cascade in fat is not well-understood and likely multifactorial, relating to adipocyte hypertrophy and dysfunction, oxidative stress, toxic lipolysis, and deficient neovascular remodeling (17). Adipose activation and adipocytokine overproduction might have systemic consequences. For example, we demonstrated over-expression of MMP-9, which plays a key role in matrix turnover and remodeling as part of the activated tissue profile. Plasma metalloproteinase concentrations are increased in obese subjects and clinical studies show that fat is a significant source (5). Because MMP-9 destabilizes atherosclerotic plaques, consequences of weight gain might be germane to cardiovascular risk (18).

Adipose tissue macrophages largely originate from circulating monocytes (13). They can exist in at least 2 differentially activated states characterized as M1 macrophages that produce pro-inflammatory cytokines linked to insulin resistance and atherosclerosis and alternative M2 phenotypes involved in immunosuppressive functions (19). In obese animals, these cell lines show predominantly M1 characteristics (20), whereas human fat displays mixed phenotypes (5). Their precise role in human disease is unknown, and whether macrophage polarization influences the pathogenic profile of fat remains an open question (5). Most probably, immune changes in humans are dynamic, with activation of pro-inflammatory “danger signals” in early phases of weight gain followed by adaptive remodeling, because the expression for many acute phase cytokines was similar in both obese groups. We demonstrated increased expression for M2 markers of CD163 and CD206, suggesting that compensatory immunosuppressive pathways might be triggered in later stages of obesity.

Our seminal finding was that obese individuals without pro-inflammatory adipose changes displayed more favorable clinical characteristics. This subset represented approximately 30% of our obese cohort, strikingly similar in proportion to metabolically healthier obese phenotypes in large population studies (21). Both WC and the presence of CLS in adipose tissue were independent predictors of brachial artery FMD, suggesting that both “quantity” and “quality” of fat might be germane to systemic disease (22).

Study limitations

To minimize subject discomfort, we relied on a single subcutaneous biopsy site for adipose tissue characterization; therefore, it remains possible that some individuals were miscategorized owing to sampling error. Our analyses were limited to subcutaneous fat, which was readily accessible. Because visceral depots are felt to be more metabolically active and pathogenic in nature, it is possible that stronger correlations could have been observed (23,24). Most participants in the present study were women, reflecting general clinical practice and sex differences in populations that seek weight loss treatments (25). Macronutrient intake plays an important role in adaptive immune responses. Although we did not specifically record daily food logs, we acknowledge that chronic differences in dietary patterns could influence metabolic states (26,27). These limitations are counterbalanced by the relatively large sample size for this type of invasive clinical study and novel translational information generated in severe human obesity where limited information currently exists.

Conclusions

We identified a group of obese subjects with reduced adipose inflammation that exhibit intermediate risk factor profiles. We hypothesize that individuals prone to inflammatory activation with weight gain might have increased cardiometabolic risk. Therapeutic modulation of the adipose phenotype might represent a novel target for treatment in obesity.

Footnotes

This work was supported by National Institutes of Health grants to Dr. Gokce (R01 HL074097 and HL084213). Dr. Vita is supported by National Institutes of Health grants HL083269, HL083801, HL081587, and HL75795. Dr. Apovian receives consulting fees from NovoNordisk, Arena, Merck, Amylin, GI Dynamics, Johnson & Johnson, Sanofi-Aventis, Orexigen, and Pfizer; and grant support from Amylin, Sanofi-Aventis, Pfizer, Orexigen, Metaproteomics, Atkins Foundation, and Arena. All other authors have reported that they have no relationships to disclose.

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(2009) Increase in plasma endotoxin concentrations and the expression of Toll-like receptors and suppressor of cytokine signaling-3 in mononuclear cells after a high-fat, high-carbohydrate meal: implications for insulin resistance. Diabetes Care32:2281–2287.

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